Use of Methionine Synthase Inhibitors for the Treatment of Fungal Diseases of Crops

ABSTRACT

The invention relates to the use of methionine synthase inhibitors for the treatment of fungal diseases of crops. The invention further relates to methods for treatment of crops against fungal diseases comprising the application of a methionine synthase inhibitor also methods for the identification of novel fungicidal compounds comprising a step for identification of methionine synthase inhibitors.

The present invention relates to the use of methionine synthaseinhibitors for the treatment of fungal diseases, and more particularlythe treatment of fungal diseases of crop plant species.

Fungi are responsible for devastating epidemics which can result insubstantial losses of crops of various plant species. The principle ofemploying inhibitors of enzymes of pathogenic fungi, and of using theseenzymes in tests in order to identify new molecules that are activeagainst these fungi, are known per se. However, merely characterizing afungal enzyme is not sufficient to achieve this objective, the enzymechosen as a target for potential antifungal molecules must also beessential to the life of the fungus, its inhibition by the antifungalmolecule resulting in death of the fungus, or essential to thepathogenesis of the fungus, in which case its inhibition is not lethalfor the fungus but merely inhibits its pathogenic capacity. Theidentification of metabolic pathways and enzymes essential to thepathogenesis and to the survival of the fungus is therefore necessaryfor the development of novel antifungal products.

The sulfur assimilation pathway comprises incorporation of the sulfateion (SO₄ ²⁻), activation thereof, and reduction thereof to reducedsulfur (S²⁻). These steps are catalyzed successively by an ATPsulfurylase (EC 2.7.7.4), an APS kinase (EC 2.7.1.25), a PAPS reductase(EC 1.8.4.8) (APS reductase in photosynthetic organisms, EC 1.8.4.9),and an (NADPH 2) sulfite reductase (EC 1.8.1.2) (a ferredoxin-dependentenzyme in photosynthetic organisms, EC 1.8.7.1). In all autotrophicorganisms, the sulfate ion assimilation, activation and reductionpathway is conserved in terms of its general principle; theincorporation of the reduced sulfur into a carbon backbone exhibitsconsiderable variations according to the organisms: bacteria¹ (forexample: Escherichia coli), plants² (for example: Arabidopsis thaliana),yeasts (for example: Saccharomyces cerevisiae ³) and filamentous fungi⁴.In fact, in plants and bacteria, the reduced sulfur is incorporated intoa molecule at C3 which derives from serine, to form cysteine. The sulfuris then transferred to a molecule at C4 which derives from homoserine,to form homocysteine. This series of reactions forms the directtranssulfuration pathway. Conversely, in Saccharomyces cerevisiae (S.cerevisae), the sulfur is directly incorporated into a molecule at C4which derives from homoserine, to form homocysteine (directsulfhydrylation)³. Cysteine is then synthesized from the homocysteine bymeans of a series of reactions which make up the reversetranssulfuration pathway. In filamentous fungi, the synthesis ofhomocysteine is carried out both by the direct pathway in plants (directtranssulfuration) and by that of S. cerevisiae (direct sulfhydrylation).Furthermore, the synthesis of cysteine is carried out either by means ofserine or from homocysteine via the reverse transsulfuration pathway.These various metabolic pathways were defined following thecharacterization of mutants auxotrophic for cysteine and for methioninein Neurospora crassa (N. crassa)⁵ and Aspergillus nidulans (A.nidulans)⁶. This model can be extrapolated to all filamentous fungi,including pathogenic fungi of plants (for example, Magnaporthe grisea,M. grisea) and of animals (for example, Aspergillus fumigatus (A.fumigatus)). M. grisea, an ascomycete-type pathogen responsible forconsiderable damage on rice crops, is a model of choice for such anapproach. Methionine synthesis in filamentous fungi requires the actionof a methionine synthase of vitamin B12-independent type as in plants.The approach described in the study of the methionine synthase gene ofCryptococcus neoformans ²⁸, a human pathogen, differs from the presentinvention. In fact, while animals (including humans) are capable ofsynthesizing methionine, this step is catalyzed by a vitaminB12-dependent type methionine synthase very different from that of theother eukaryotes such as plants and fungi. The plant methionine synthaseexhibits strong homologies at the protein level with that of M. grisea,but also exhibits structural-type differences according to the modelingcarried out^(9,12,27). Thus, identification of the fungal enzyme andcharacterization thereof are required in order to determine its specificcharacteristics, allowing the identification of solely fungalinhibitors. The choice and the application of such inhibitors in methodsfor treating plant crops will then be specific. Thus, the presentinvention describes the fact that the mutants of the MET6 gene, and moreparticular the deletion mutants of the MET6 gene encoding the methioninesynthase of M. grisea are auxotrophic for methionine and arenonpathogenic. In these mutants, the infectious process is greatlyeffected at the level of the phase of penetration of the pathogen intothe plant cell, but also in terms of its ability to progress in theinfected tissues. The pathogenic capacity of the M. grisea methioninesynthase mutants is partially restored when methionine is added duringinfection. These results show that the absence of methionine synthaseactivity is lethal to the fungus during infection. Similar results havebeen obtained in Utilago maydis (U. maydis) and Phytophthora infestans(P. infestans).

DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID No. 1: Magnaporthe grisea methionine synthase gene-   SEQ ID No. 2: Magnaporthe grisea methionine synthase cDNA-   SEQ ID No. 3: Magnaporthe grisea methionine synthase protein    sequence-   SEQ ID No. 4: Met6-5 primer-   SEQ ID No. 5: Met6-6 primer-   SEQ ID No. 6: HphRP10 primer-   SEQ ID No. 7: Met6-7 primer-   SEQ ID No. 8: Met6-10 primer-   SEQ ID No. 9: dCGS-hph-end(−) primer-   SEQ ID No. 10: Met6-8 primer-   SEQ ID No. 11: Met6-9 primer-   SEQ ID No. 12: Met6-1 primer-   SEQ ID No. 13: Met6-2 primer-   SEQ ID No. 14: Met6-3 primer-   SEQ ID No. 15: Met6-4 primer-   SEQ ID No. 16: U. maydis methionine synthase gene-   SEQ ID No. 17: U. maydis methionine synthase cDNA-   SEQ ID No. 18: U. maydis methionine synthase protein sequence-   SEQ ID No. 19: P. infestans methionine synthase EST sequence-   SEQ ID No. 20: deduced P. infestans methionine synthase protein    sequence.

DESCRIPTION OF THE INVENTION

A subject of the present invention is methods for treating crops againstfungal diseases by application of an effective amount of a methioninesynthase inhibitor.

In the context of the present invention, the fungal diseases are definedas diseases due to pathogenic plant fungi belonging to the ascomycete,basidiomycete and oomycete families.

A subject of the invention is a method for controlling, in a curative orpreventive capacity, phytopathogenic fungi of crops, characterized inthat an effective (agronomically effective) and nonphytotoxic amount ofa methionine synthase inhibitor is applied to the soil where the plantsgrow or are liable to grow, to the leaves and/or the fruit of the plantsor to the seeds of the plants. The term “effective and nonphytotoxicamount” is intended to mean an amount of inhibitor that is sufficient toallow the control of the developmental cycle or the destruction of thefungi which are present or which may appear on the crops, and that doesnot result in any notable symptom of phytotoxicity for said crops. Suchan amount may vary within broad limits depending on the fungal family tobe controlled, the type of crop, the climatic conditions and thecompounds included in the antifungal composition according to theinvention.

This amount can be determined by systematic field trials, which arewithin the scope of those skilled in the art.

The methods according to the invention are of use for treating the seedsof cereals (wheat, rye, triticale and barley, in particular), potato,cotton, pea, rapeseed, maize or flax, alternatively the seeds of foresttrees or else genetically modified seeds of these plants. The presentinvention also relates to foliar application to plant crops i.e. to thefoliage, the leaves, the fruit and/or the stems of the plants concerned,but also any other type of application. Among the plants targeted by themethods according to the invention, mention may be made of rice, maize,cotton, cereals, such as wheat, barley or triticale, fruit trees, inparticular apple trees, pear trees, peach trees, vine, banana trees,orange trees, lemon trees, etc., oil-yielding crops, for examplerapeseed or sunflower, market garden and vegetable crops, tomatoes,salads, protein-yielding crops, pea, Solanaceae, for example potato,beetroot, flax, and forest trees, and also genetically modified homologsof these crops.

Among the plants targeted by the method according to the invention,mention may be made of:

wheat, as regards controlling the following seed diseases: fusaria(Microdochium nivale and Fusarium roseum), stinking smut (Tilletiacaries, Tilletia controversa or Tilletia indica), septoria disease(Septoria nodorum); loose smut (Ustilago tritici);

wheat, as regards controlling the following diseases of the parts of theplant above ground: cereal eyespot (Tapesia yallundae, Tapesiaacuiformis), take-all (Gaeumannomyces graminis), foot blight (F.culmorum, F. graminearum), head blight (F. culmorum, F. graminearum,Microdochium nivale), black speck (Rhizoctonia cerealis, powdery mildew(Erysiphe graminis form a specie tritici), rusts (Puccinia striiformisand Puccinia recondita) and septoria diseases (Septoria tritici andSeptoria nodorum), net blotch (Drechslera tritici-repentis);

barley, as regards controlling the following seed diseases: net blotch(Pyrenophora graminea, Pyrenophora teres and Cochliobolus sativus),loose smut (Ustilago nuda) and fusaria (Microdochium nivale and Fusariumroseum);

barley, as regards controlling the following diseases of the parts ofthe plant above ground: cereal eyespot (Tapesia yallundae), net blotch(Pyrenophora teres and Cochliobolus sativus), powdery mildew (Erysiphegraminis form a specie hordei), dwarf leaf rust (Puccinia hordei) andleaf blotch (Rhynchosporium secalis);

potato, as regards controlling tuber diseases (in particularHelminthosporium solani, Phoma tuberosa, Rhizoctonia solani, Fusariumsolani), and mildew (Phytophthora infestans);

potato, as regards controlling the following foliage diseases: earlyblight (Alternaria solani), mildew (Phytophthora infestans);

cotton, as regards controlling the following diseases of young plantsgrown from seeds: damping-off and collar rot (Rhizoctonia solani,Fusarium oxysporum), black root rot (Thielaviopsis basicola);

protein-yielding crops, for example pea, as regards controlling thefollowing seed diseases: anthracnose (Ascochyta pisi, Mycosphaerellapinodes), fusaria (Fusarium oxysporum), gray mold (Botrytis cinerea),mildew (Peronospora pisi);

oil-yielding crops, for example rapeseed, as regards controlling thefollowing seed diseases: Phoma lingam, Alternaria brassicae andSclerotinia sclerotiorum;

maize, as regards controlling seed diseases: (Rhizopus sp., Penicilliumsp., Trichoderma sp., Aspergillus sp. and Gibberella fujikuroi);

flax, as regards controlling seed diseases: Alternaria linicola;

forest trees, as regards controlling damping-off (Fusarium oxysporum,Rhizoctonia solani);

rice, as regards controlling the following diseases of the parts aboveground: blast disease (Magnaporthe grisea), black speck (Rhizoctoniasolani);

vegetable crops, as regards controlling the following diseases of seedsor of young plants grown from seeds: damping-off and collar rot(Fusarium oxysporum, Fusarium roseum, Rhizoctonia solani, Pythium sp.);

vegetable crops, as regards controlling the following diseases of theparts above ground: gray mold (Botrytis sp.), powdery mildews (inparticular Erysiphe cichoracearum, Sphaerotheca fuliginea, Leveillulataurica), fusaria (Fusarium oxysporum, Fusarium roseum), leaf spot(Cladosporium sp.), alternaria leaf spot (Alternaria sp.), anthracnose(Colletotrichum sp.), septoria leaf spot (Septoria sp.), black speck(Rhizoctonia solani), mildews (for example, Bremia lactucae, Peronosporasp., Pseudoperonospora sp., Phytophthora sp.);

fruit trees, as regards diseases of the parts above ground: moniliadisease (Monilia fructigenae, M. laxa), scab (Venturia inaequalis),powdery mildew (Podosphaera leucotricha);

grapevine, as regards foliage diseases: in particular, gray mold(Botrytis cinerea), powdery mildew (Uncinula necator), black rot(Guignardia biwelli), mildew (Plasmopara viticola);

beetroot, as regards the following diseases of the parts above ground:cercosporia blight (Cercospora beticola), powdery mildew (Erysiphebeticola), leaf spot (Ramularia beticola).

Methionine synthase is a well characterized enzyme that is found inplants and microorganisms (bacteria, yeasts, fungi). The methods of thepresent invention use methionine synthase inhibitors. In a firstembodiment, the invention relates to the use of inhibitors of fungalmethionine synthase, more preferably of inhibitors of the methioninesynthase of a phytopathogenic fungus, for the treatment of fungaldiseases of crops.

Preferably, the methionine synthase is isolated, purified or partiallypurified from its natural environment. The methionine synthase can beprepared by means of various methods. These methods are in particularpurification from natural sources such as cells that naturally expressthese polypeptides, production of recombinant polypeptides byappropriate host cells and subsequent purification thereof, productionby chemical synthesis or, finally, a combination of these variousapproaches. These various methods of production are well known to thoseskilled in the art.

In one of the embodiments of the invention, the methionine synthase ispurified from an organism that naturally produces this enzyme, forinstance bacteria such as E. coli, yeasts such as S. cerevisiae, orfungi such as N. crassa or M. grisea.

In a preferred embodiment of the invention, the methionine synthase isoverexpressed in a recombinant host organism. The methods of engineeringDNA fragments and the expression of polypeptides in host cells are wellknown to those skilled in the art and have, for example, been describedin “Current Protocols in Molecular Biology” Volumes 1 and 2, Ausubel F.M. et al., published by Greene Publishing Associates andWiley-Interscience (1989) or in Molecular Cloning, T. Maniatis, E. F.Fritsch, J. Sambrook (1982).

In a specific embodiment of the invention, the methionine synthaseinhibitors inhibit the methionine synthase of M. grisea, of U. maydis,and more particularly represented by a sequence comprising the sequenceidentifier SEQ ID No. 18, or else of P. infestans, in particularrepresented by a sequence comprising the sequence identifier SEQ ID No.20; said methionine synthase can be encoded by the gene of M. grisearepresented by a sequence comprising the sequence identifier SEQ ID No.1, or by the cDNA represented by a sequence comprising the sequenceidentifier SEQ ID No. 2, by the gene of U. maydis represented by asequence comprising the sequence identifier SEQ ID No. 16, or by thecDNA represented by a sequence comprising the sequence identifier SEQ IDNo. 17, or else by the gene of P. infectans represented by a sequencecomprising the sequence identifier SEQ ID No. 19.

A subject of the present invention is also antifungal compositionscomprising a methionine synthase inhibitor and another antifungalcompound. Mixtures with other antifungal compounds are particularlyadvantageous, especially mixtures with acibenzolar-5-methyl,azoxystrobin, benalaxyl, benomyl, blasticidin-S, bromuconazole,captafol, captan, carbendazim, carboxin, carpropamide, chlorothalonil,antifungal compositions based on copper or on copper derivatives such ascopper hydroxide or copper oxychloride, cyazofamide, cymoxanil,cyproconazole, cyprodinyl, dichloran, diclocymet, dicloran,diethofencarb, difenoconazole, diflumetorim, dimethomorph, diniconazole,discostrobin, dodemorph, dodine, edifenphos, epoxyconazole, ethaboxam,ethirimol, famoxadone, fenamidone, fenarimol, fenbuconazole, fenhexamid,fenpiclonil, fenpropidine, fenpropimorph, ferimzone, fluazinam,fludioxonil, flumetover, fluquinconazole, flusilazole, flusulfamide,flutolanil, flutriafol, folpet, furalaxyl, furametpyr, guazatine,hexaconazole, hymexazol, imazalil, iprobenphos, iprodione,isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb,mefenoxam, mepanipyrim, metalaxyl and its enantiomers such asmetalaxyl-M, metconazole, metiram-zinc, metominostrobin, oxadixyl,pefurazoate, penconazole, pencycuron, phosphoric acid and itsderivatives such as fosetyl-Al, phthalide, picoxystrobin, probenazole,prochloraz, procymidone, propamocarb, propiconazole, pyraclostrobin,pyrimethanil, pyroquilon, quinoxyfen, silthiofam, simeconazole,spiroxamine, tebuconazole, tetraconazole, thiabendazole, thifluzamide,thiophanate, e.g. thiophanate-methyl, thiram, triadimefon, triadimenol,tricyclazole, tridemorph, trifloxystrobin, triticonazole, valinamidederivatives, for instance iprovalicarb, vinclozolin, zineb and zoxamide.The mixtures thus obtained have a broader spectrum of activity. Thecompositions according to the invention can also comprise one or moreinsecticides, bactericides, acaricides or pheromones, or other compoundsthat have a biological activity.

A subject of the present invention is also methods for producing anantifungal composition using a methionine synthase inhibitor.

A subject of the present invention is also methods for preparingantifungal compounds, comprising the identification of compounds whichinhibit the enzymatic activity of methionine synthase.

The enzymatic reaction is carried out in the presence of the testcompound in order to measure the inhibition of the enzymatic activity ofthe methionine synthase. All biochemical tests for measuring theenzymatic activity of methionine synthase and therefore identifyingcompounds which inhibit this enzymatic activity can be used in themethods according to the invention.

A high-throughput biochemical assay is proposed in order to screen forspecific inhibitors of this enzyme.

Preferably, the methods for identifying compounds which inhibit theactivity of methionine synthase comprise bringing these compounds intocontact with methionine synthase in the presence of its substrates:homocysteine, methyl tetrahydrofolate or polyglutamate derivatives ofmethyl tetrahydrofolate ((CH₃—H₄)PteGlu_(n)), and of various cofactorssuch as phosphate, magnesium and zinc; and measuring the enzymaticactivity.

Measuring the enzymatic activity of methionine synthase can beassociated with measuring the formation of methionine, oftetrahydrofolate or else of methenyl tetrahydrofolate or of any productthus obtained, or else measuring said activity by any other chemical orenzymatic reaction.

The measurement of the enzymatic activity of methionine synthase canalso be carried out in the presence of a coupling enzyme.S-Adenosylmethionine synthase (AdoMetS) can be used as such; itcatalyzes the formation of S-adenosylmethionine (S-AdoMet) in thepresence of methionine, ATP and magnesium. The measurement of theenzymatic activity of methionine synthase can then be associated withthe measurement of the formation of S-adenosylmethionine, of phosphateor of pyrophosphate.

According to another aspect of the invention, the methods foridentifying compounds which inhibit the enzymatic activity of methioninesynthase comprise expressing methionine synthase in a host organism,purifying the methionine synthase produced by the host organism,bringing these compounds into contact with the purified methioninesynthase and its substrates, and measuring the enzymatic activity.

In a preferred embodiment, all these methods comprise an additional stepin which it is determined whether said compounds which inhibit theenzymatic activity of methionine synthase inhibit fungal growth and/orpathogenesis.

The present invention therefore relates to methods for identifyingcompounds which inhibit fungal growth and/or pathogenesis by inhibitingthe enzymatic activity of methionine synthase. These methods consist insubjecting a compound, or a mixture of compounds, to an appropriateassay for identifying the compounds which inhibit methionine synthase,and in selecting the compounds which react positively to said assay,where appropriate in isolating them, and then in identifying them.

Preferably, the appropriate assay is an assay of the enzymatic activityof methionine synthase as defined above.

Preferably, a compound identified according to these methods issubsequently tested for these antifungal properties according to methodsknown to those skilled in the art. Preferably, the compound is evaluatedby means of phenotypic tests such as pathogenesis assays on detachedleaves or on whole plants.

The term “compound” is intended to mean, according to the invention, anychemical compound or mixture of chemical compounds, including peptidesand proteins.

The term “mixture of compounds” is understood to mean, according to theinvention, at least two different compounds, such as, for example, the(dia)stereoisomers of a molecule, mixtures of natural origin derivedfrom the extraction of biological material (plants, plant tissues,bacterial cultures, cultures of yeasts or of fungi, insects, animaltissues, etc.) or reaction mixtures that have not been purified or havebeen completely or partially purified, alternatively mixtures ofproducts derived from combinatorial chemistry techniques.

Finally, the present invention relates to novel fungalpathogenesis-inhibiting compounds which inhibit the enzymatic activityof methionine synthase, in particular the compounds identified by meansof the methods according to the invention and/or the compounds derivedfrom the compounds identified by means of the methods according to theinvention.

Preferably, the fungal pathogenesis-inhibiting compounds which inhibitthe enzymatic activity of methionine synthase are not general inhibitorsof enzymes. Also preferably, the compounds according to the inventionare not compounds already known to have an antifungal activity and/or anactivity on fungal pathogenesis.

EXAMPLE 1 Characterization of the Methionine Synthase Gene in Fungi

The methionine synthase gene was identified in the genome of M. griseaversion V2 using the protein sequence of the methionine synthase of A.nidulans ⁷ (NCBI, accession number: AAF82115) as model. The completenucleotide sequence of the methionine synthase gene located on Contig2.150 (MG_contig_(—)2.150, position 6196-8629, complementary strand, SEQID No. 1) comprises 3 exons corresponding to a cDNA of 2301 bp (SEQ IDNo. 2) which encodes a polypeptide of 766 amino acids (SEQ ID No. 3).The sequence of the g ene and the splicing resulting in the definitivemessenger could be confirmed by virtue of the numerous ESTs identifiedin the various public and private bases. The M. grisea methioninesynthase is encoded by a single gene as in A. nidulans ⁷. Analysis ofthe primary protein sequence deduced from the putative cDNA shows from48% to 79% homology with the vitamin B₁₂-independent methioninesynthases of S. cerevisae (P05694), of A. nidulans (AAF82115), of thebacterium E. coli (P13009) and of the plant A. thaliana (AAF00639).

The primary sequence of the M. grisea methionine synthase has twoconserved domains corresponding to the methionine synthase domain (334residues, E=4e⁻¹¹⁶, pfam01717) characteristic of this enzyme. Thisdomain allows the production of methionine by transfer of a methyl groupfrom methyl tetrahydrofolate triglutamate to homocysteine. This regionis located in the C-terminal part of the protein. A second domain,COG0620 or methionine synthase II (methyltransferase) concerns theN-terminal part of the protein (330 amino acids)⁸. It has recently beenpossible to determine the specificity of each of these domains withrespect to the substrates of the enzyme, homocysteine and methyltetrahydrofolate, and to the reaction product, methionine, on the enzymecrystallized from A. thaliana ⁹.

The primary sequence of the M. grisea methionine synthase was used tosearch for orthologs in the various fungal species whose genome ispartially or completely sequenced. These various primary sequences weresubsequently compared with the methionine synthases described in variousorganisms such as plants, bacteria and animals. The characterization ofthe structure of the genes (introns+exons) and of the primary amino acidsequences was carried out using the appropriate programs (tblastn;FGENSH; PSI-PHI-BLAST). According to this procedure, methionine synthasecould be characterized in several fungi (ascomycetes and basidiomycetes)and a phytopathogenic oomycete (P. sojae and P. infestans). Aphylogenetic tree for methionine synthase could be established and therepresentation obtained shows that the M. grisea methionine synthasebelongs to the methionine synthases of ascomycetes and that it isdistant from those of basidiomycetes. Overall, the tree obtained is inagreement with that which retraces the phylogenetic origin of theseorganisms¹⁰.

EXAMPLE 2 Deletion of the Magnaporthe grisea Methionine Synthase Gene

The study of the role of the methionine synthase gene in the developmentand the infectious process of M. grisea was carried out by studying thephenotype of deletion mutants of this gene. The strategy for obtainingdeletion mutants is based on replacing the MET6 gene with a mutantallele in which the MET6 open reading frame has been replaced with acassette for resistance to an antibiotic for selection of thetransformants (hygromycin).

The construction of this vector for replacing the M. grisea MET6 gene iscarried out in two steps: (i) PCR amplification of the regions whichborder this gene and which correspond respectively to genomic regions ofapproximately 1 kb located on either side of MET6, (ii) ligation ofthese genomic DNA fragments to a gene for resistance to an antibioticthat makes it possible to select the transformants. Thus, the PCRfragments used to replace the gene consist of the two regions which arereferred to as left border and right border of the gene studied (FIG.1). We selected the hygromycin resistance gene (HYG, comprising thePtrpC promoter, the coding portion of the hygromycin resistance genehph) as selectable gene. Ligation of the HYG gene is first carried outvia the SacII/BglII sites and the EcoRI/SacII sites of the left border(Met6-1/Met6-2 primers) of the MET6 gene. The right border of the MET6gene (Met6-3/Met6-4 primers) is then introduced via the PmeI sitedownstream of the hph gene (FIG. 1). The replacement vector thereforecomprises the left border (BG) of the MET6 gene (promoter region of 1475bp), the HYG cassette (1400 bp) and the right border (BD) of the MET6gene (terminator region of 1251 bp). The ligation product (BG-hph-BD)was subsequently cloned into a plasmid vector. The MET6 gene replacementcassette was subsequently amplified from the corresponding plasmid byPCR with primers specific for the ends of the borders of the MET6 gene(Met6-5/Met6-6 primers). Sequencing of the junctions between the bordersand the HYG cassette of the replacement vector made it possible toverify the construction. The PCR product (1 μg), purified by agarose gelelectrophoresis, is then used to transform protoplasts of the M. griseaP1.2 wild-type strain according to conventional techniques developed inthe laboratory. The products derived from the transformation areselected on a medium containing the corresponding antibiotic(hygromycin).

EXAMPLE 3 Identification and Trophic Characterization of the met6Δ::hphDeletion Mutants Obtained by Gene Replacement

The primary transformants are selected for their ability to develop inthe presence of hygromycin. The identification of the met6Δ::hph mutantsis carried out by measuring the differential growth of the transformantson a minimum medium containing hygromycin, supplemented or notsupplemented with 1 mM methionine. The met6Δ::hph mutants are incapableof developing on this minimum medium, but they have a normal growth onthis minimum medium supplemented with methionine. The frequency of themutants is of the order of 20% of the primary transformants analyzedunder our experimental conditions. These mutants were subsequentlygenetically purified by isolation of monosphores.

The five met6Δ::hph mutants obtained (4.1, 15.1, 22.1 and 23.1) areincapable of developing on a minimum medium that allows growth of theP1.2 wild-type strain. The addition of methionine to the minimum mediumrestores growth of the mutants. The trophic complementation of themet6Δ::hph mutants by the addition of methionine indicates that themethionine synthase is affected by the MET6 gene deletion.

The addition of sulfur donors such as cysteine or glutathione(precursors of homocysteine, a substrate from methionine synthase) isnot sufficient to restore the growth of the M. grisea met6 Δ::hphmutants. Consequently, there is not parallel pathway which would usemethyl tetrahydrofolate or its polyglutamate derivatives for thesynthesis of methionine in this fungus. Thus, the de novo synthesis ofmethionine is catalyzed solely by methionine synthase. The activity ofthis enzyme is therefore essential to the development of the fungus.

On the other hand, in the presence of S-adenosylmethionine (SAM orAdoMet), a compound which derives directly from methionine and which isessential to the cell cycle, or of S-methylmethionine (SMM), a compoundwhich is synthesized in plants, the M. grisea met6Δ::hph mutants arecapable of developing, although with a reduced growth compared to theP1.2 wild-type strain. SAM and SMM are capable of penetrating and ofbeing metabolized to methionine by M. grisea. This mechanism is probablysimilar to that described in the yeast S. cerevisae. In fact, in thisascomycete fungus,

SAM and SMM are incorporated into the cell via the transporters SAM3 andMPP1, and are subsequently converted to methionine in the presence ofhomocysteine by homocysteine-5-methyltransferases (SAM4 and MHT1) whichuse SAM or SMM, respectively, as methyl group donor (S. cerevisae)¹¹.According to our experimental conditions, the addition of SAM (1 mM) tothe minimum medium is more effective than that of SMM in restoring thegrowth of the met6Δ::hph mutants. These results suggest that M. griseahas transporters and homocysteine methyltransferases similar to thosedescribed in S. cerevisae. An analysis of the M. grisea genome bysequence homology search (tBlastN) with respect to the yeast SAM4 andMHT1 proteins makes it possible to demonstrate a gene which is anortholog of SAM4 in M. grisea. It would appear that filamentous fungihave only one gene (no ortholog of MHT1). According to our results(better growth on SAM than on SMM), this protein could have a greateraffinity for SAM than for SMM.

Several “ectopic” transformants, corresponding to transformants whichhave integrated the replacement vector BG-hph-BD at a locus other thanthat of the MET6 gene, were also analyzed. These hygromycin-resistantectopic transformants are capable of developing on a minimum medium. Themethionine synthase gene is therefore functional in these ectopictransformants and the vector has been inserted at a locus of the genomewhich has no effect on the development of the pathogen under ourexperimental conditions (ability to develop on the minimum or completemedium, sporulation).

EXAMPLE 4 Molecular Characterization of the met6Δ::hph Mutants

The met6Δ::hph mutants are cultured in a medium containing methionine (1mM) in order to extract the genomic DNA, which will be used to perform amolecular analysis of the MET6 locus by PCR and by Southernhybridization.

The molecular analysis of the transformants is carried out byamplification of the genomic regions of the MET6 locus using the variousspecific primers for replacing the wild-type allele of the MET6 gene ofP1.2 with the mutant allele met6Δ::hph. These PCRs are carried out foreach mutant with specific oligonucleotides. The reactions useoligonucleotides which hybridize: firstly, with the hygromycinresistance gene hph and, secondly, with a genomic sequence of the MET6locus located outside the MET6 region used to construct the replacementvector (left junction and right junction); with the sequences homologousto the MET6 gene. Thus, the amplification of a fragment of 1969 bp (leftjunction) or of 2447 bp (right junction) occurs only in the case ofreplacement of the wild-type gene with the met6Δ::hph gene of theconstruct (primers HphRP01rev (SEQ ID No. 6)/Met6-7(−) (SEQ ID No. 7)and Met6-10 (SEQ ID No. 8)/dCGS-hph-end(−) (SEQ ID No. 9) (FIG. 1)). Noamplification is obtained in the case of the P1.2 wild-type strain or ofthe ectopic transformants. Similarly, the absence of amplification ofthe MET6 gene in the met6Δ::hph mutants (primers Met6-8, SEQ ID No.10/Met6-9, SEQ ID No. 11) (FIG. 1) is an indication that the MET6 geneis indeed absent in these transformants. On the other hand, with theseprimers, a fragment of 2424 bp is amplified in the P1.2 wild-type strainand in the ectopic transformants. Only the PCRs carried out with themutants 22.2 and 23.1 give the expected results with these three typesof PCR. No amplification could be obtained, for unexplained reasons,with the mutants 4.1 and 15.1 in the PCRs Met6-10 (SEQ ID No.8)/dCGS-hph-end (SEQ ID No. 9) and hphRP01 (SEQ ID No. 6)/Met6-7(−), SEQID No. 7).

An analysis by Southern hybridization after digestion of the genomic DNAwith the BamH1 restriction enzyme was carried out and the hybridizationsignals obtained for the 4 mutants were compared with those obtainedwith the P1.2 wild-type strain and the ectopic transformant (19.1).Using the (Met6-1 (SEQ ID No. 12)/Met6-2 (SEQ ID No. 13)) PCR fragmentcorresponding to the MET6 left border present in the replacement vector(MET6 promoter region) as probe, 2 bands are observed for the mutants,the sizes of which are different from those of the P1.2 wild-type strainand of the ectopic transformant (19.1). The signal corresponds to theMET6 promoter region in the P1.2 wild-type strain and the ectopictransformant 19.1. The latter also shows a hybridization signalcorresponding to the replacement vector inserted into another genomicregion. A similar result is obtained using a (Met6-3 (SEQ ID No.14)/Met6-4 (SEQ ID No. 15)) PCR fragment corresponding to the MET6 rightborder present in the replacement vector (MET6 terminator region). Witha probe specific for the inserted gene (hph), only the met6Δ::hphmutants and the ectopic transformant 19.1 show a hybridization signalcorresponding either to the presence of hph at the MET6 locus (mutants)or to the replacement vector BG-met6Δ::hph-BD (ectopic). The latterresults indicate that the various mutants analyzed are identical at themolecular level and contain just one copy of the hph gene inserted asthe MET6 locus in place of the MET6 coding phase.

EXAMPLE 5 Analysis of the Pathogenic Capacity of the Magnaporthe griseamet6Δ::hph Mutants

The pathogenic capacity of the M. grisea met6 Δ::hph mutants wasevaluated by means of an infection test on barley leaves under survivalconditions and on whole barley and rice plants. This analysis wasfollowed by measurement of the spore germination rate, of appressorialdifferentiation and of penetration into barley leaves. The spores of theP1.2 wild-type strain and of the met6Δ::hph mutants 4.1, 15.1, 22.1,23.1 and 24.1 are harvested after growth for 14 days on a riceflour-based medium containing 1 mM. The plant material used is barley(cv. Express) and rice (cv. Sariceltik).

In the experiments carried out on barley leaves under survivalconditions, the leaves are incubated on an agar medium (1% agar-H₂O)containing kinetin (2 mg.ml⁻¹) in a temperate climatic chamber (26° C.)at high humidity (100%) and under light of 100 microeinsteins. Duringinfection, the spores are deposited onto the leaves either in the formof droplets (35 μl) or by coating the surface of the leaves with thesuspension of spores using a cotton wool bud. These experiments arecarried out in the absence or in the presence of 1 mM methioninethroughout the incubation or for only 24 hours. The spore concentrationis 3×10⁴ spores/ml to 1×10⁵ spores/ml in water. The appearance of thesymptoms caused by M. grisea is then observed for at least 7 days ofincubation. Inoculation of barley leaves under survival conditions, withspores of the M. grisea wild-type strain P1.2, causes necrosescharacteristic of the development of the fungus in the infected leaf(sporulating lesions). Conversely, the met6Δ::hph mutants 4.1, 15.1,22.1, 23.1 and 24.1 do not cause any symptom on the barley leaves,regardless of the method of inoculation, and are thereforenonpathogenic. The addition of methionine to the met6Δ::hph mutantspores makes it possible to partially restore their pathogenic capacity(development of characteristic but nonsporulating lesions). Furthermore,under our experimental conditions, we did not observe any markeddifferences between the infections carried out with injured ornoninjured leaves. These observations suggest that the met6Δ::hphmutants are incapable of penetrating barley leaves, even injured barleyleaves. The yellowing of the leaves observed in the experiments carriedout with spreading of the spores by coating is probably due to prematureageing of the leaves under our experimental conditions. The results aresummarized in the following table:

TABLE I Estimation of the pathogenic capacity of the Magnaporthe griseamet6Δ::hph mutants with respect to barley Barley leaves under survivalconditions - Magnaporthe grisea 3 × 10⁴ spores/ml Without With 1 mMmethionine methionine Wild-type strains P1.2 +++ +++ Ectopic mutantstrains Ectopic 19.1 +++ +++ Ectopic 20.1 +++ +++ met6Δ::hph mutants 4.1 0 + 15.1 0 + 22.1 0 + 23.1 0 + Legend: 0: no lesions; +:nonsporulating necroses (lesions); +++: sporulating lesions

The observation and the quantification of the various steps of theinfection (germination, formation of the appressorium and penetration)are carried out based on an inoculation of barley leaves under survivalconditions with drops of 35 μl of a suspension of spores (3×10⁵spores/ml). After 24 hours, the epidermis of the leaf is peeled in orderto observe, under the microscope, the number of germinated spores thathave differentiated an appressorium and the number of events ofpenetration into the epidermal cell. A solution of calcofluor at 0.01%makes it possible to cause an intense fluorescence of the walls of thefungal cells located at the surface of the plant (the hyphae located inthe epidermal cell are not colored). These observations (Table II)demonstrate that the met6Δ::hph mutants have a slightly reducedgermination (−10% to −40% compared to the wild-type strain). Theirappressorial differentiation rate is also slightly reduced (0% to −30%compared to the wild-type strain). On the other hand, these mutantappressoria are incapable of penetrating into the foliar tissues.

TABLE II Development of the Magnaporthe grisea met6Δ::hph mutants onbarley % appressorium/ M. grisea % germinated % strains germinationspores penetration ΔMET6-4.1 45 52 0 ΔMET6-15.1 70 66 ND ΔMET6-22.1 6280 ND ΔMET6-23.1 37 63 0 Wild-type P1.2 80 80 100 Ectopic 19.1 80 80 100Ectopic 20.1 80 80 ND ND: not determined

The formation of appressoria is also observed under an artificialcondition where the spores are germinated on teflon membranes with orwithout the addition of methionine (1 mM concentration under ourexperimental conditions). These very hydrophobic membranes mimic thesurface of the leaf, thereby making it possible to induce the formationof appressoria and to readily measure the appressorial differentiationrate.

In conclusion, the M. grisea met6Δ::hph mutants are therefore incapableof penetrating into the plant, although they differentiate appressoria.These results show that these mutants have nonfunctional appressoria.

Pots containing 3-week-old barley plants (corresponding to the emergenceof the second leaf) are subjected to spraying with a suspension of M.grisea spores (10 ml of water containing spores at the concentration of3×10⁴ spores/ml and 0.3% gelatin for adhesion of the water droplets tothe leaves). The observations of the symptoms are carried out for atleast 8 days after spraying. The barley plants treated with thewild-type strain (P1.2) and the ectopic transformants (19.1 and 20.1)show necrotic lesions caused by the development of the fungus in theinfected leaves. On the other hand, no symptom of disease was observedin the case of the inoculation with the M. grisea met6Δ::hph mutants.These mutants are therefore considered to be nonpathogenic. Furthermore,this study indicates, like the analysis carried out with the barleyleaves under survival conditions, that the amount of methionine (or ofother compounds that derive from methionine, such as SAM and SMM) in thetissues of the leaf is insufficient to complement the deficiency inmethionine synthesis of the met6Δ::hph mutants. Thus, the pathwaydemonstrated in the in vitro experiments (complementation in thepresence of SAM and of SMM) does not appear to be functional in thefungus while it is growing in the plant.

The met6Δ::hph mutants obtained correspond to the deletion of the codingphase of the MET6 gene which is replaced by the hph gene, conferringhygromycin resistance. These mutants are incapable of synthesizingmethionine from homocysteine and are therefore incapable of multiplyingon a minimum medium. The addition of methionine to this minimum mediumis essential for the development of these mutants. The met6Δ::hphmutants do not cause any symptom of disease when they are used toinoculate barley leaves either under survival conditions, or wholeplants, even at high spore concentrations. Thus, methionine synthesis bymethionine synthase is essential to the development of the fungus, bothin vitro and in planta.

The met6Δ::hph mutants can differentiate an appressorium in the absenceof methionine. This aspect indicates that the spore can possess a notinsignificant store of methionine allowing synthesis of proteins andmetabolites necessary for the development of this cell. This store mustcome from the methionine that was supplied to the mutant in order toallow its growth and its sporulation on the rice flour-based medium used(containing 1 mM of methionine). On the other hand, the absence ofpenetration of the met6Δ::hph mutants into the leaves indicates thatsaid mutants differentiate nonfunctional appressoria incapable ofdirecting penetration of the fungus into the plant. It is probable thatthe mutant appressoria have rapidly exhausted their methionine stores.In fact, the addition of methionine to the mutant spores allowspenetration into the plant and the beginning of development in the leaf.However, said development is not complete and the absence of formationof sporulating lesions suggests that, once the methionine store suppliedto the spores is exhausted, the plant is incapable of covering themethionine needs of the mutants.

EXAMPLE 6 Methods for Assaying and Characterizing Molecules whichInhibit the Enzymatic Activity of Methionine Synthase

The method involves the characterization of all molecules whose actioninhibits the consumption of substrates or the formation of productsdetermined according to direct or indirect techniques (which include theuse of a “coupling” enzyme for measuring the activity of methioninesynthase). Methionine synthase catalyzes an irreversible reaction in thepresence of homocysteine, of methyl tetrahydrofolate (n=1) or of itspolyglutamate derivatives (n≧3) in the presence of various cofactors(phosphate, magnesium, zinc) according to known assays described in theliterature^(12,13,14,15,27). The methodology includes determiningmethionine or the folate derivatives produced, by techniques forseparating compounds by reverse phase HPLC chromatography¹⁶. Theassaying of the tetrahydrofolate produced during the reaction after ithas been converted to methenyl tetrahydrofolate can be carried out on aspectrophotometer at 350 nm, since the methyl tetrahydrofolate substrateis not detectable under the experimental conditions described in theprocedure¹⁷. A proposed alternative is to assay the methionine synthaseactivity in the presence of S-adenosylmethionine synthetase. In thisassay, the S-adenosylmethionine synthetase (AdoMetS) of M. grisea willpreferably be used, but any of the AdoMetS can be used as a “coupling”enzyme. The AdoMetS enzyme catalyzes the irreversible reaction which, inthe presence of methionine, ATP and magnesium, producesS-adenosylmethionine (SAM), phosphate and pyrophosphate.

The methionine synthase activity is assayed, in the end, through theamount of SAM, of phosphate or of pyrophosphate produced, by means of acolorimetric method and/or on a spectrophotometer after conversion ofthe products in the presence of a coupling enzyme or by means of anyother chemical or enzymatic reaction for measuring methionine synthaseactivity. These various methodologies are the subject of manydescriptions in the literature and can be adapted according to theexperimenter^(18,19,20,21,22,23). For example, the sensitivity of themethod for assaying methionine synthase activity in the presence of SAMsynthetase can be improved through the addition of a pyrophosphatasewhich converts the pyrophosphate into 2 mol of phosphate. Thus, for eachmole of methionine synthesized, the method produces 3 mol of phosphate.

Purification of Magnaporthe grisea Methionine Synthase

The production of a large amount of methionine synthase is carried outusing techniques that use expression vectors for overproduction of theprotein in bacteria or yeast. The technique preferably uses cloning ofthe cDNA into an expression vector which makes it possible to integratea His-Tag extension at the N-terminal or C-terminal end of the protein.For example, when the pET-28b(+) vector (Novagen)²⁴ is selected, the2301 bp cDNA is cloned into NdEI and EcoRI according to conventionalmolecular biology techniques. The construct obtained, calledpET-28-MgMET6, is introduced into the Escherichia coli BL21 type DE3(pLysS) bacterial strain and the expression is produced after inductionwith IPTG (0.5 mM). The recombinant bacteria are cultured at 28° C. for4 hours. The cells are then harvested by centrifugation and the pelletobtained is resuspended in lysis buffer suitable for the stability ofthe protein. After sonication of the preparation, the soluble fractioncontaining the recombinant protein, obtained after centrifugation, isloaded onto a column of Ni-NTA agarose type. The purification and theelution of the enzyme are then carried out after several successivewashes of the matrix with an imidazole solution. The procedure followsthe protocol define by Qiagen²⁵.

After elution, the protein fraction containing the recombinantmethionine synthase is concentrated by ultrafiltration and subjected tomolecular filtration on PD10 (Pharmacia)²⁷ in order to remove all tracesof imidazole. The purification of the recombinant protein can beaccompanied by a second step consisting of molecular filtration bychromatography on Superdex S200 (Pharmacia)²⁶ or of ion exchangechromatography on MonoQ HR10/10 (Pharmacia)²⁶. The activity of themethionine synthase is followed during the purification, using theappropriate direct measurement assay.

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1. A method for treating crops against fungal diseases, characterized inthat an effective amount of a methionine synthase inhibitor is applied.2. The method as claimed in claim 1, characterized in that themethionine synthase is of fungal origin.
 3. The method as claimed inclaim 2, characterized in that the methionine synthase is derived fromMagnaporthe grisea.
 4. The method as claimed in claim 3, in which themethionine synthase comprises SEQ ID No.
 3. 5. The method as claimed inclaim 3, in which the methionine synthase is encoded by a sequencecomprising SEQ ID No. 1 or SEQ ID No.
 2. 6. The method as claimed inclaim 2, characterized in that the methionine synthase is derived fromUstilago maydis.
 7. The method as claimed in claim 6, in which themethionine synthase comprises SEQ ID No.
 18. 8. The method as claimed inclaim 6, in which the methionine synthase is encoded by a sequencecomprising SEQ ID No. 16 or SEQ ID No.
 17. 9. The method as claimed inclaim 2, characterized in that the methionine synthase is derived fromPhytophthora infestans.
 10. The method as claimed in claim 9, in whichthe methionine synthase comprises SEQ ID No.
 20. 11. The method asclaimed in claim 9, in which the methionine synthase is encoded by asequence comprising SEQ ID No.
 19. 12. The method as claimed in claim 1,in which the methionine synthase inhibitor is in the form of anantifungal composition.
 13. An antifungal composition, characterized inthat it comprises a methionine synthase inhibitor and an antifungalcompound.
 14. A method for producing an antifungal composition,characterized in that a methionine synthase inhibitor is used.
 15. Amethod for preparing antifungal compounds, comprising the identificationof compounds which inhibit the enzymatic activity of methioninesynthase.
 16. The method as claimed in claim 15, in which theidentification of compounds which inhibit the enzymatic activity ofmethionine synthase comprises the following steps: bringing saidcompound into contact with methionine synthase in the presence ofhomocysteine and of methyl tetraglutamate or its polyglutamatederivatives, and of cofactors; and measuring said enzymatic activity.17. The method as claimed in claim 16, in which the identification ofcompounds which inhibit the enzymatic activity of methionine synthasecomprises the following steps: bringing said compound into contact withmethionine synthase in the presence of homocysteine and ofmethyltetraglutamate or its polyglutamate derivatives, and of cofactors,and measuring the formation of methionine.
 18. The method as claimed inclaim 17, in which the identification of compounds which inhibit theenzymatic activity of methionine synthase comprises the following steps:bringing said compound into contact with methionine synthase in thepresence of homocysteine, of methyl tetrahydrofolate and of phosphate,magnesium and zinc, and measuring the formation of methionine. 19.Method according to claim 15, in which the identification of compoundswhich inhibit the enzymatic activity of methionine synthase comprisesthe following steps: bringing said compound into contact with methioninesynthase in the presence of homocysteine, of methyl tetrahydrofolate orits polyglutamate derivatives, of S-adenosylmethionine synthetase, ofATP and of Mg, and of cofactors, and measuring the formation ofS-adenosylmethionine, phosphate or pyrophosphate.
 20. The method asclaimed in claim 19, in which the identification of compounds whichinhibit the enzymatic activity of methionine synthase comprises thefollowing steps: bringing said compound into contact with methioninesynthase in the presence of homocysteine, of methyl tetrahydrofolate orits polyglutamate derivatives, of S-adenosylmethionine synthetase, ofATP and of Mg, and of cofactors, and measuring the formation ofphosphate.
 21. The method as claimed in claim 15, in which theidentification of compounds which inhibit the enzymatic activity ofmethionine synthase comprises the following steps: expressing methioninesynthase in a host organism; purifying the methionine synthase producedby said host organism; bringing said compound into contact with saidpurified methionine synthase in the presence of homocysteine and ofmethyl tetrahydrofolate or its polyglutamate derivatives, and ofcofactors; and measuring the enzymatic activity.
 22. The method asclaimed in claim 21, in which the identification of compounds whichinhibit the enzymatic activity of methionine synthase comprises thefollowing steps: expressing methionine synthase in a host organism;purifying the methionine synthase produced by said host organism;bringing said compound into contact with said purified methioninesynthase in the presence of homocysteine, of methyl tetrahydrofolate andof phosphate, magnesium and zinc, and measuring the formation ofmethionine.
 23. The method as claimed in claim 15, in which theidentification of compounds which inhibit the enzymatic activity ofmethionine synthase comprises the following steps: expressing methioninesynthase in a host organism; purifying the methionine synthase producedby said host organism; bringing said compound into contact with saidpurified methionine synthase in the presence of homocysteine, of methyltetrahydrofolate or its polyglutamate derivatives, ofS-adenosylmethionine synthetase, of ATP and of Mg, and of cofactors, andmeasuring the formation of S-adenosylmethionine, of phosphate or ofpyrophosphate.
 24. The method as claimed in claim 15, in which themethionine synthase is of fungal origin.
 25. The method as claimed inclaim 24, in which the methionine synthase which is expressed is encodedby a polynucleotide selected from the sequences comprising SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 16, SEQ ID No. 17 and/or SEQ ID No.
 19. 26. Themethod as claimed in claim 24, in which the methionine synthase which isexpressed is selected from the sequences comprising SEQ ID No. 3, SEQ IDNo. 18 and/or SEQ ID No.
 20. 27. The method as claimed in claim 15,comprising an additional step in which it is determined whether saidcompounds which inhibit the enzymatic activity of MS inhibit fungalgrowth and/or pathogenesis.
 28. A methionine synthase inhibitoridentified by means of one of the methods as claimed in claim 15.